Controlled release compositions of estradiol metabolites

ABSTRACT

The present invention provides improved sustained release formulations of estradiol metabolites, including 2-hydroxyestradiol, 2-methoxyestradiol, 4-hydroxyestradiol and 4-methoxyestradiol, useful for therapeutic treatments. The invention also provides methods of producing sustained release forms of estradiol metabolites. The compositions of the present invention include microparticles, nanoparticles, patches, crystals, gels, rods, stints, pallets, discs, lozenges, wafers, capsules, films, microcapsules nanocapsules, hydrogels, liposomes, implants and vaginal rings. Compositions also include formulations for transdermal and intravenous delivery of estradiol metabolites. The present invention provides numerous improvements over previous forms of estradiol metabolites, such advantages including the sustained release of normally short half-life compounds to maintain therapeutic blood levels.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority from U.S. Provisional PatentApplication No. 60/377,490 filed May 3, 2002 entitled, “EstradiolMetabolites and Ester Derivatives of Estradiol Metabolites,” whichapplication is hereby incorporated by this reference in its entirety

FIELD OF THE INVENTION

This invention relates generally to sustained release forms of estradiolmetabolites, as well as methods of making and using them.

BACKGROUND OF THE INVENTION

Estradiol is converted into different derivatives through metabolicprocesses in vivo. Two particular types of metabolites are thecatecholestrogens and the methoxyestradiols. The catecholestrogens,2-hydroxyestradiol and 4-hydroxyestradiol, are created by hydroxylationof estrogen via cytochrome P450 enzymes. The catecholestrogens can bemethylated by catechol-O-methyl-transferase to create themethoxyestradiols, 2-methoxyestradiol and 4-methoxyestradiol.

Estradiol metabolites have been reported to have an effect on a numberof cellular processes. They apparently inhibit angiogenesis and thepolymerization and organization of tubulin in actively growing cells andinduce apoptosis in some cells. In addition, 2-hydroxyestradiol and2-methoxyestradiol appear to affect cholesterol levels in ovariectomizedrats and to inhibit adipose cell proliferation in culture, while2-hydroxyestradiol and 2-methoxyestradiol apparently decreases theeffects of obesity, metabolic syndrome, and vascular and renaldysfunction in obese rats. Estradiol metabolites are also reported to bebeneficial in the treatment of end-stage renal disease and asthma.Additionally, estradiol metabolites appear to be effective antifungalagents.

Beneficial effects of estradiol metabolites have also been reported forcancer treatment. 2-methoxyestradiol appears to decrease the growth oflung cancer cells in culture when administered with wild-type p53, toinhibit the growth of human pancreatic and prostate cancer cells and tobe toxic to osteosarcoma cells. 2-methoxyestradiol was also reported todecrease the growth rate of neuroblastoma cells and tumors of thepituitary gland. Estradiol metabolites also apparently increase theintracellular accumulation of superoxide anions in rapidly dividingcells and enhance the effects of existing cancer treatments, such asradioimmunotherapy.

Thus, estradiol metabolites may be useful in the treatment or preventionof a variety of diseases. Unfortunately, naturally occurring estradiolmetabolites have poor bioavailability and a short half-life, and thebeneficial effects appear to be tied to a prolonged period of treatment.A need exists for pharmaceutical formulations of estradiol metabolites,which increase the duration of action of the metabolites withoutnecessitating frequent administrations, which would be undesirable inboth animal and human patients. The development of a sustained releasesystem for estradiol metabolites would provide an improved therapeuticoption for treatment of a wide-variety of veterinary and human diseases.

SUMMARY OF THE INVENTION

In accordance with the purpose(s) of this invention, as embodied andbroadly described herein, this invention, in one aspect, relates tosustained release formulations of estradiol metabolites and methods ofmaking and using the same.

In certain embodiments, the compositions and methods may comprise anestradiol metabolite and a material providing for sustained release.Such material providing for sustained release may be selected from thegroup consisting of microparticles, nanoparticles, patches, crystals,gels, rods, stints, pellets, discs, lozenges, wafers, capsules, films,microcapsules, nanocapsules, hydrogels, liposomes, implants and vaginalrings. In addition, the invention further provides for hydrophilicpolymers. In certain embodiments, the present invention providescompositions of matter or methods utilizing prodrugs of estradiolmetabolites. Such prodrugs may be ester derivatives of estradiolmetabolites. In other embodiments, the estradiol metabolite may bederivatized.

Hydrophilic polymers of use in the present invention may include, butare not limited to, poly(ethylene glycol), poly(propylene glycol) andcopolymers of poly(ethylene glycol) and poly(propylene glycol).

In a particular embodiment, estradiol metabolites are catecholestrogensor methoxyestradiols. In particular embodiments, they are selected fromthe group consisting of 2-methoxy estradiol, 2-hydroxy estradiol,4-methoxy estradiol and 4-hydroxy estradiol.

In other particular embodiments, microparticles or nanoparticles maycomprise a biodegradable polymer selected from the group consisting ofpoly(lactide)s, poly(glycolide)s, poly(lactide-co-glycolide)s,polyoactic acid)s, poly(glycolic acid)s, poly(lactic acid-co-glycolicacid)s, polycaprolactone, polycarbonates, polyesteramides,polyanhydrides, poly(amino acids), polyorthoesters, polyacetyls,polycyanoacrylates, polyetheresters, poly(dioxanone)s, poly(alkylenealkylate)s, copolymers of polyethylene glycol and polyorthoester,biodegradable polyurethanes, blends and copolymers thereof.

Estradiol metabolites of use in the present invention may be selectedfrom the group consisting of 2-methoxy estradiol, 2-hydroxy estradiol,4-methoxy estradiol and 4-hydroxy estradiol. In a certain embodiment,the ester derivative of an estradiol metabolite is selected from thegroup consisting of 3-benzoyl-2-methoxy estradiol; 17-benzoyl-2-methoxyestradiol; 17-acetyl-2-methoxy estradiol; 3-acetyl-2-methoxy estradiol;3,17-dibenzoyl-2-methoxy estradiol; 3,17-diacetyl-2-methoxy estradiol;3-benzoyl-4-methoxy estradiol; 17-benzoyl-4-methoxy estradiol;17-acetyl-4-methoxy estradiol; 3-acetyl-4-methoxy estradiol;3,17-dibenzoyl-4-methoxy estradiol; 3,17-diacetyl-4-methoxy estradiol;3-benzoyl-2-hydroxy estradiol; 17-benzoyl-2-hydroxy estradiol;17-acetyl-2-hydroxy estradiol; 3-acetyl-2-hydroxy estradiol;3,17-dibenzoyl-2-hydroxy estradiol; 3,17-diacetyl-2-hydroxy estradiol;2,3-dibenzoyl-2-hydroxy estradiol; 2,17-dibenzoyl-2-hydroxy estradiol;2,17-diacetyl-2-hydroxy estradiol; 2,3-diacetyl-2-hydroxy estradiol;2,3,17-tribenzoyl-2-hydroxy estradiol; 2,3,17-triacetyl-2-hydroxyestradiol; 34)enzoyl-4-hydroxy estradiol; 17-benzoyl-4-hydroxyestradiol; 17-acetyl-4-hydroxy estradiol; 3-acetyl-4-hydroxy estradiol;3,17-dibenzoyl-4-hydroxy estradiol; 3,17-diacetyl-4-hydroxy estradiol;3,4-dibenzoyl-4-hydroxy estradiol; 4,17-dibenzoyl-4-hydroxy estradiol;4,17-diacetyl-4-hydroxy estradiol; 3,4-diacetyl-4-hydroxy estradiol;3,4,17-tribenzoyl-4-hydroxy estradiol; 3,4,17-triacetylthydroxyestradiol.

Derivatives include but are not limited to dicarboxylic acid compounds,diacids, polar compounds and ionic compounds.

The compositions of the present invention may be applied transdermally,such as by buccal, oral, ocular, nasal, rectal or vaginal application.The compositions and methods of the present invention may also utilizeestradiol metabolites in a eutectic mixture.

The compositions and methods of the present invention may utilizesustained release forms of estradiol metabolites produced by any methodknown in the art. In particular embodiments, such production methodswill include but are not limited to, spray drying a solution of polymerand estradiol metabolite dissolved in an organic solvent; wetemulsification including a continuous and discontinuous phase followedby solvent removal; selective extraction of an oil phase solvent andemulsion. Any of the production methods may further provide an annealingstep.

The compositions and methods of the present invention may be useful totreat an individual.

Additional advantages of the invention will be set forth in part in thedescription which follows, and in part will be obvious from thedescription, or may be learned by practice of the invention. Theadvantages of the invention will be realized and attained by means ofthe elements and combinations particularly pointed out in the appendedclaims. It is to be understood that both the foregoing generaldescription and the following detailed description are exemplary andexplanatory only and are not restrictive of the invention, as claimed.

DESCRIPTION OF THE INVENTION

The present invention may be understood more readily by reference to thefollowing detailed description of particular embodiments of theinvention and the Examples included therein.

Before the present compounds, compositions, and/or methods are disclosedand described, it is to be understood that this invention is not limitedto specific synthetic methods, specific reagents or to laboratory ormanufacturing techniques, as such may, of course, vary, unless it isotherwise indicated. It is also to be understood that the terminologyused herein is for the purpose of describing particular embodiments onlyand is not intended to be limiting.

DEFINITIONS

For the purposes of the present invention, the following terms shallhave the following meanings:

For the purposes of the present invention, the term “biodegradable”refers to polymers that dissolve or degrade in vivo within a period oftime that is acceptable in a particular therapeutic situation. This timeis typically less than five years and usually less than one year afterexposure to a physiological pH and temperature, such as a pH rangingfrom 6 to 9 and a temperature ranging from 25° C. to 38° C.

The term “analog” and its cognates refer to any molecule thatdemonstrates estradiol metabolite activity. Such molecule may be asynthetic analog, fragment of estradiol metabolite or endogenousbiological molecule other than an estradiol metabolite capable ofestradiol metabolite-like activity. In sum, an estradiol metaboliteanalog refers to any molecule that demonstrates bioactivity similar orgreater than an estradiol metabolite itself.

For the purposes of the present invention, the term “prodrug” refers toany modification of an estradiol metabolite, including a physical orchemical alteration that results in an increased plasma circulationtime, increased encapsulation efficiency and/or increased watersolubility. The chemical modification or modifications to the drug arereversible upon administration to an individual by endogenousmechanisms.

The product of such endogenous mechanisms is an estradiol metabolite.

For the purposes of the present invention, the term “estradiolmetabolite” includes any molecule that results from the metabolicbreakdown of estradiol; and any molecule derived from an estradiolmetabolite, such as a prodrug, or an analog.

For the purposes of the present invention, the term “drug” may refer toany estradiol metabolite, any estradiol metabolite analog, or estradiolmetabolite prodrug.

Moreover, for the purposes of the present invention, the term “a” or“an” entity refers to one or more of that entity; for example, “aprodrug” or “an estradiol metabolite molecule” refers to one or more ofthose compounds or at least one compound. As such, the terms “a” or“an”, “one or more” and “at least one” can be used interchangeablyherein. It is also to be noted that the terms “comprising,” “including,”and “having” an be used interchangeably. Furthermore, a compound“selected from the group consisting of” refers to one or more of thecompounds in the list that follows, including mixtures (i.e.combinations) of two or more of the compounds.

For the purpose of the present invention, a “eutectic” mixture iscomposed of two or more substances that melt at the lowest possibletemperature.

According to the present invention, an isolated or biologically puremolecule is a compound that has been removed from its natural milieu. Assuch, “isolated” and “biologically pure” do not necessarily reflect theextent to which the compound has been purified. An isolated compound ofthe present invention can be obtained from its natural source, can beproduced using laboratory synthetic techniques or can be produced by anysuch chemical synthetic route.

For the purposes of the present invention, ranges may be expressedherein as from “about” one particular value, and/or to “about” anotherparticular value. When such a range is expressed, another embodimentincludes from the one particular value and/or to the other particularvalue. Similarly, when values are expressed as approximations, by use ofthe antecedent “about,” it will be understood that the particular valueforms another embodiment. It will be further understood that theendpoints of each of the ranges are significant both in relation to theother endpoint, and independently of the other endpoint.

Finally, for the purposes of the present invention, the term“individual” means an animal or human of either gender.

Reference will now be made in detail to particular embodiments of theinvention.

Naturally occurring estradiol metabolites have a short plasma half-life.Oral bioavailability is low, in part due to rapid hepatic metabolism. Inaddition, some indications that can be treated using estradiolmetabolites, estradiol metabolite analogs, or estradiol metaboliteprodrugs, such as diabetic nephropathy or obesity, require prolongedadministration of the drug(s). Development of estradiol metabolites,estradiol metabolite analogs, or estradiol metabolite prodrugs dosageforms for delivery over extended time periods is a novel way toadminister these particular therapeutics in a useful formulation.

Two main strategies exist to prolong the duration of exposure to rapidlymetabolized drugs, particularly steroids. The first is to increaseplasma circulation time by chemically modifying the steroid with organicacids to form steroid ester prodrugs. After delivery, the steroid esterbond is cleaved to form the parent compound by endogenous enzymes.Physical and chemical properties imparted to the steroid by the organicacid, or other modifying compound, govern the rate at which the parentcompound is released from its prodrug form. In this way, the plasmacirculation time can be increased in a controlled manner. Sustainedexposure to a steroid ester prodrug may be realized by any deliveryroute, including intravenous, peroral, intramuscular, subcutaneous,transdermal, rectal, ocular, and so on. Certain embodiments of thepresent invention provide compositions for water-soluble formulations ofestradiol metabolites, estradiol metabolite analogs, or estradiolmetabolite prodrugs with increased half-lives that may be deliveredparenterally or orally.

The second strategy to achieve sustained exposure to estradiolmetabolite therapeutics is to incorporate estradiol metabolites,estradiol metabolite analogs, or estradiol metabolite prodrugs into somepolymeric sustained release delivery system. Sustained release devicesmay incorporate estradiol metabolites, estradiol metabolite analogs, orestradiol metabolite prodrugs, themselves with increased plasmahalf-lives over that of the parent compounds. Of particular advantage inthe manufacturing of sustained release delivery systems incorporatingestradiol metabolites is the ability to modify the physical and chemicalproperties of the metabolite, such as by esterification, in order tooptimize the release properties of the final delivery system.

In certain embodiments of the present invention, formulations mayconsist of a mixture of estradiol metabolites, estradiol metaboliteanalogs, or estradiol metabolite prodrugs and another compound thatforms a liquid at body temperature, such as in an eutectic mixture. Inother embodiments, formulations are described that consist of areservoir and permeation aids for the transdermal delivery of estradiolmetabolites, estradiol metabolite analogs or estradiol metaboliteprodrugs thereof. In yet other embodiments, the invention describes thecomposition of polymer matrices for the controlled release of estradiolmetabolites, estradiol metabolite analogs, or estradiol metaboliteprodrugs thereof. Such matrices may be composed of polymermicroparticles or nanoparticles. In particular embodiments, suchpolymers are biodegradable.

Estradiol Metabolites

In certain embodiments, the estradiol metabolite is a catecholestradiolsuch as 2-hydroxyestradiol (Estra-1,3,5(10)-triene-2,3,17-triol (17β))or 4-hydroxyestradiol (Estra-1,3,5(10)-triene-3,4,17-triol (17β)) or amethoxyestradiol, such as 2-methoxyestradiol(Estra-1,3,5(10)-triene-2-methoxy-3,17-diol (17β)) or 4-methoxyestradiol(Estra-1,3,5(10)-triene-4-methoxy-3,17-diol (17β)). Commercialpreparations of all of these compounds are readily available. Inaddition, a method of producing a highly purified 2-methoxyestradiol isdisclosed in U.S. Provisional Patent Application No. 150,293.

In certain embodiments, the estradiol metabolite may be attached to ahydrophilic polymer. The hydrophilic polymer may be selected from thegroup consisting of poly(propylene glycol), poly(ethylene glycol),copolymers of poly(ethylene glycol) and poly(propylene glycol). Inparticular embodiments the hydrophilic molecule is poly(ethylene glycol)(PEG).

In an alternative embodiment, the estradiol metabolite is associatedwith microparticles or nanoparticles. In certain embodiments, themicroparticles or nanoparticles selected from the group consisting ofpoly(lactide)s, poly(glycolide)s, poly(lactide-co-glycolide)s,poly(lactic acid)s, poly(glycolic acid)s, poly(lactic acid-co-glycolicacid)s, polycaprolactone, polycarbonates, polyesteramides,polyanhydrides, poly(amino acids), polyorthoesters, polyacetyls,polycyanoacrylates, polyetheresters, poly(dioxanone)s, poly(alkylenealkylate)s, copolymers of polyethylene glycol and polyorthoester,polyurethanes, blends and copolymers thereof. In a particularembodiment, the microparticle or nanoparticle ispoly(lactide-co-glycolide) (PLGA). In another particular embodiment, themicroparticle or nanoparticle is composed of a biodegradable polymer.

The skilled artisan will realize that the compounds listed above areexemplary only and that many variations may be used, depending on theparticular hydroxylation or methylation site on the parent estradiolcompound. For example, estradiol can be hydroxylated or methylated atmany sites and such variations are known in the art.

Esters of Estradiol Metabolites

In certain embodiments, esters of estradiol metabolites are utilized tocreate prodrugs. The ester linkage stays intact during preparation andstorage of the drug, only becoming vulnerable to hydrolysis afteradministration to a patient. Therefore, esters are optimal prodrugsbecause a physiological environment has abundant endogenous esterases tocatalyze hydrolysis of the linkage. Once hydrolysis occurs, only theactive estradiol metabolite and a non-toxic biological compound remain,such as acetic acid or propionic acid, for example.

In a certain embodiment, esters of estradiol metabolites are utilized tocontrol solubility of the estradiol metabolites. Water solubility may beconferred by esterifying with succinic acid, for example. Other estersmay improve solubility in a variety of other solvents, and may alsoallow some interaction between an estradiol metabolite ester and somepolymer comprising a sustained release delivery device, which, in turn,would control the release of the ester prodrug from the polymer matrixand into the surrounding tissue fluids.

In a particular embodiment, esters of 2-methoxyestradiol include, butare not limited to 3-benzoyl-2-methoxyestradiol,17-benzoyl-2-methoxyestradiol 17-acetyl-2-methoxyestradiol,3-acetyl-2-methoxyestradiol, 3,17-benzoyl-2-methoxyestradiol and3,17-diacetyl-2-methoxyestradiol.

In another particular embodiment esters of 4-methoxyestradiol include,but are not limited to 3-benzoyl-4-methoxyestradiol,17-benzoyl-methoxyestradiol, 17-acetyl-4-methoxyestradiol,3-acetyl-4-methoxyestradiol 3,17-benzoyl-4-methoxyestradiol and3,17-diacetyl-4-methoxyestradiol.

In an alternate particular embodiment, esters of 2-hydroxyestradiolinclude, but are not limited to, 3-benzoyl-2-hydroxyestradiol,17-benzoyl-2-hydroxyestradiol, 17-acetyl-2-hydroxyestradiol,3-acetyl-2-hydroxyestradiol, 3,17-dibenzoyl-2-hydroxyestradiol,3,17-diacetyl-2-hydroxyestradiol, 2,3-dibenzoyl-2-hydroxyestradiol,2,17-dibenzoyl-2-hydroxyestradiol, 2,17-diacetyl-2-hydroxyestradiol,2,3-diacetyl-2-hydroxyestradiol, 2,3,17-tribenzoyl-2-hydroxyestradioland 2,3,17-triacetyl-2-hydroxyestradiol.

In another particular embodiment, esters of 4-hydroxyestradiol include,but are not limited to, 3-benzoyl hydroxyestradiol,17-benzoyl-4-hydroxyestradiol, 17-acetyl-4-hydroxyestradiol,3-acetyl-4-hydroxyestradiol, 3,17-dibenzoyl-4-hydroxyestradiol,3,17-diacetyl-4-hydroxyestradiol, 3,4-dibenzoyl-4-hydroxyestradiol,4,17-dibenzoyl-4-hydroxyestradiol, 4,17-diacetyl-4-hydroxyestradiol,3,4-diacetyl-4-hydroxyestradiol, 3,4,17-tribenzoyl-4-hydroxyestradioland 3,4,17-triacetyl-4-hydroxyestradiol.

In a certain embodiment, esters of all four estradiol metabolites may beorganic acid derivatives of the original estradiol metabolite.Particular embodiments include, but are not limited to, esters ofpropionic acid, butyric acid, valeric acid, hexanoic acid, benzoic acid,acetic acid, propionic acid, butyric acid, stearic acid and other fattyacids.

Estradiol metabolites of use in the present invention may be selectedfrom the group consisting of 2-methoxyestradiol, 2-hydroxyestradiol,4-methoxyestradiol and 4-hydroxyestradiol. In a particular embodiment,the ester derivative of an estradiol metabolite is selected from thegroup consisting of 3-benzoyl-2-methoxyestradiol;17-benzoyl-2-methoxyestradiol; 17-acetyl-2-methoxyestradiol;3-acetyl-2-methoxyestradiol; 3,17-dibenzoyl-2-methoxyestradiol;3,17-diacetyl-2-methoxyestradiol; 3-benzoyl-4-methoxyestradiol;17-benzoyl-4-methoxyestradiol; 17-acetyl-4-methoxyestradiol;3-acetyl-4-methoxyestradiol; 3,17-dibenzoyl-4-methoxyestradiol;3,17-diacetyl-4-methoxyestradiol; 3-benzoyl-2-hydroxyestradiol;17-benzoyl-2-hydroxyestradiol; 17-acetyl-2-hydroxyestradiol;3-acetyl-2-hydroxyestradiol; 3,17-dibenzoyl-2-hydroxyestradiol;3,17-diacetyl-2-hydroxyestradiol; 2,3-dibenzoyl-2-hydroxyestradiol;2,17-dibenzoyl-2-hydroxyestradiol; 2,17-diacetyl-2-hydroxyestradiol;2,3-diacetyl-2-hydroxyestradiol; 2,3,17-tribenzoyl-2-hydroxyestradiol;2,3,17-triacetyl-2-hydroxyestradiol; 3-benzoyl-4-hydroxyestradiol;17-benzoyl-4-hydroxyestradiol; 17-acetyl-4-hydroxyestradiol;3-acetyl-4-hydroxyestradiol; 3,17-dibenzoyl-4-hydroxyestradiol;3,17-diacetyl-4-hydroxyestradiol; 3,4-dibenzoyl-4-hydroxyestradiol;4,17-dibenzoyl-4-hydroxyestradiol; 4,17-diacetyl-4-hydroxyestradiol;3,4-diacetyl-4-hydroxyestradiol; 3,4,17-tribenzoyl-4-hydroxyestradiol;3,4,17-triacetyl-4-hydroxyestradiol.

Methods for synthesizing several esters of estradiol metabolites areknown. (See, e.g., Japanese Patent NO. 57,041,479 and 49,100,070).

In an alternative embodiment, the ester of an estradiol metabolite maybe attached to a hydrophilic polymer. A hydrophilic polymer increasesthe half-life of the compound and also allows for less frequent andlower dose administrations. In certain embodiments, a hydrolysablelinkage is included to free the ester molecule from the hydrophilicpolymer after hydrolysis. This will allow the ester molecule to enterthe cytoplasm of cells only after hydrolysis as it is slower or unableto pass through a cell membrane with a hydrophilic molecule, such asPEG, attached.

In certain embodiments, the ester of an estradiol metabolite, with orwithout a hydrophilic polymer attached, may also be incorporated inmicroparticles, such as microspheres or nanospheres.

The skilled artisan will realize that the compounds listed above areexemplary only and that many variations may be used, depending on theparticular ester derivative created from a particular estradiolmetabolite. Such variations are known in the art.

Preparation of Intravenous or Oral Formulations of Estradiol Metabolites

In certain embodiments, estradiol metabolites, estradiol metaboliteanalogs, or estradiol metabolite prodrugs are derivatized with polar orionic compounds, such that the derivative is water soluble and exhibitsprolonged plasma circulation times compared to the parent metabolite. Ina particular embodiment, estradiol metabolites, estradiol metaboliteanalogs, or estradiol metabolite prodrugs are derivatized withdicarboxylic acid compounds, including but not limited to oxalic,malonic, maleic, succinic, glutaric, adipic, pimelic, pamoic or otherdiacids. In another particular embodiment, diacids with shorterintervening carbon chains, such as succinic, glutaric, maleic, malonic,or oxalic acids are used. Compounds such as these confer increased watersolubility and increased plasma circulation times when combined withestradiol metabolites. Methods of esterification with diacids may beeffected with the appropriate anhydride or mixed anhydride of the diacidusing techniques well known in the art. The invention includes allmodifications to estradiol metabolites, estradiol metabolite analogs, orestradiol metabolite prodrugs that achieve increased water solubilityand plasma lifetime. Such derivatives may be conceived and syntheticpathways for such derivatives may be readily executed by those withordinary skill in the art (e.g. U.S. Pat. No. 2,897,218). Any availablefunctional group on the estradiol metabolite or estradiol metaboliteester may be derivatized.

Preparation of Transdermal Formulations of Estradiol Metabolites

In certain embodiments, estradiol metabolites, estradiol metaboliteanalogs, or estradiol metabolite prodrugs thereof are mixed withappropriate vehicles comprising a reservoir from which the drug canpartition into the skin at an appropriate rate. The drug must diffusethrough the protective stratum corneum barrier before entering thedermal layer, from which systemic drug absorption takes place.Permeation may be enhanced by physical means, such as increasedhydration, application of ultrasound, thermal, or electrical potentials,or by chemical means, such as incorporation of fatty acid esters,chaotropic agents, polyols, terpenoids, or surfactants. Partitioningbetween the vehicle and stratum corneum depends on the relativesolubility of the drug in each environment. Thus, both the identity ofthe vehicle and composition of the drug, via esterification, forexample, may be changed to optimize the release profile of the drug fromthe transdermal patch. In a particular embodiment, solutions or solidsuspensions of estradiol metabolite are made in media containing skinpenetration enhancers and/or biocompatible solvent or mixtures ofsolvents or a transdermal adhesive. The suspension is designed topromote the dissolution into and transdermal permeation of the drugthrough the stratum corneum. In another particular embodiment, estradiolmetabolites, estradiol metabolite analogs, or estradiol metaboliteprodrugs are dissolved in a vehicle such as menthol, so that theformulation is liquid at skin temperature. In another particularembodiment estradiol metabolites, estradiol metabolite analogs, orestradiol metabolite prodrugs are suspended or dissolved in anappropriate transdermal adhesive and incorporated into a standard drugin adhesive transdermal patch; or incorporated into a multilayered patchcomprising drug in adhesive (next to the skin), a rate controllingmembrane, and drug in adhesive serving as a reservoir.

Preparation of Sustained Release Microparticle Formulations of EstradiolMetabolites

Hepatic first pass metabolism decreases the bioavailability of orallydelivered steroids. As a result, steroids are often given by injection.Steroids are typically short acting, and chronic treatment requiresrepeated injections. Formation of estradiol metabolite or estradiolmetabolite analog prodrugs by esterification of steroid and derivatationor parenteral delivery in an oil vehicle decrease dosing frequency. Analternative way to achieve therapeutic levels of estradiol metabolitesfor a prolonged period of time is to incorporate estradiol metabolites,estradiol metabolite analogs or estradiol metabolite prodrugs intoextended delivery devices.

A certain embodiment of the present invention provides microspherescomposed of poly-D,L-(lactide-co-glycolide). This polyester isbiocompatible, with a long record of medical safety. Polymer erosion inthe body controls the rate of drug release and one skilled in the art isadept at manipulating this rate. Extended release dosing systems are anideal compliment to estradiol metabolite treatments for conditions suchas type II diabetes, which often require lifelong drug therapy.

The invention further provides methods for encapsulating estradiolmetabolites, estradiol metabolite analogs, or estradiol metaboliteprodrugs in suitable polymeric delivery devices for sustained release.Drug may be dispersed through the polymer matrix, or alternatively, thedrug, either alone or as a mixture with another polymer, solvent, orother agent may be surrounded by a polymeric capsule. Release of thedrug may be controlled by diffusion through the polymer matrix, or by acombination of drug diffusion through the polymer matrix and erosion ofthe polymeric delivery device. Preferred devices include rods, stints,pellets, discs, lozenges, wafers, capsules, films, microparticles, ornanoparticles, microcapsules, or nanocapsules. In a particularembodiment, estradiol metabolites, estradiol metabolite analogs, orestradiol metabolite prodrugs are associated with a biodegradablepolymer in microparticle or nanoparticle form.

In certain embodiments, the estradiol metabolites, estradiol metaboliteanalogs, or estradiol metabolite prodrugs are associated with a polymerin a microparticle form. In a preferred embodiment, a microparticle hasa preferred diameter of less than 1.0 mm and is preferably between 1.0and 200.0 micrometers. Microparticles include both microspheres andmicrocapsules. Microspheres are typically solid spherical microparticlesand microcapsules are microspheres with a core of a different polymer,drug or composition.

In certain embodiments, the estradiol metabolites, estradiol metaboliteanalogs, or estradiol metabolite prodrugs, with or without a hydrophilicpolymer attached, are associated with biodegradable submicron particlesfor controlled release of the metabolite molecules. A nanoparticle has adiameter ranging from 20.0 nanometers to about 2.0 microns and istypically between 100.0 nanometers and 1.0 micron.

Nanoparticles can be created by any technique well known in the art.They can be created in the same manner as microparticles, except thathigh-speed mixing or homogenization is used to reduce the size of thepolymer/bioactive agent emulsions to less than 2.0 microns andpreferably below 1.0 micron. (See, e.g., WO 97/04747)

In certain embodiments, the microparticles or nanoparticles arecomprised of poly(lactide)s, poly(glycolide)s,poly(lactide-co-glycolide)s, poly(lactic acid)s, poly(glycolic acid)s,poly(tactic acid-co-glycolic acid)s, polycaprolactone, polycarbonates,polyesteramides, polyanhydrides, poly(amino acids), polyorthoesters,polyacetyls, polycyanoacrylates, polyetheresters, poly(dioxanone)s,poly(alkylene alkylate)s, copolymers of polyethylene glycol andpolyorthoester, biodegradable polyurethanes, blends and copolymersthereof.

In another embodiment, the microparticle or nanoparticle ispoly(lactide-co-glycolide) (PLGA). PLGA degrades when exposed tophysiological pH and hydrolyzes to form lactic acid and glycolic acid,which are normal byproducts of cellular metabolism. The disintegrationrate of PLGA polymers will vary depending on the polymer molecularweight, ratio of lactide to glycolide monomers in the polymer chain, andstereoregularity of the monomer subunits. Polymer disintegration rateswill be increased by mixtures of L and D stereoisomers that disrupt thepolymer crystallinity. In addition, microspheres may contain blends oftwo or more biodegradable polymers, of different molecular weight and/ormonomer ratio.

In other alternative embodiments, derivatized biodegradablemicroparticles, including hydrophilic polymers attached to PLGA, can beused to form microspheres.

The illustrative embodiments describe methods for the encapsulation ofestradiol metabolites, estradiol metabolite analogs, or estradiolmetabolite prodrugs. Methods may be chosen and adapted based on severalconsiderations including solubility of the estradiol metabolite in aparticular solvent, desired physical state of the drug in the finaldelivery system, desired drug loading in the microsphere deliverysystem, desired release rate and duration of release of the drug fromthe delivery system, desired particle size, and so forth. Microspherescan be made by any technique well known in the art. In certainembodiments, microspheres are produced by single or double emulsionsteps followed by solvent removal. In alternative embodiments, otherknown methods such as spray drying, solvent evaporation, phaseseparation and coacervation may be utilized to create microspheres.Other methods and variations of the above are also known in the art andmay also be used with the present invention.

In a particular embodiment, polymeric microparticles are formed by spraydrying a solution of polymer and estradiol metabolites, estradiolmetabolite analogs, or estradiol metabolite prodrugs dissolved in anappropriate organic solvent. The concentrations of the polymer andsolvent are controlled to give microparticles that contain apredetermined weight ratio of drug to polymer. The core load, in part,controls the release properties of that particular drug from thedelivery device.

In another particular embodiment, polymeric microparticles are formed bywet emulsification followed by solvent removal. Drug and polymer aredissolved in a suitable organic solvent that will comprise thediscontinuous, dispersed phase of the emulsion. In certain embodiments,the discontinuous phase solvent will also contain a preservative, suchas an antioxidant, buffer, or other agent intended to preserve thechemical integrity of the microparticle components. The preservative maybe dissolved or suspended in the discontinuous phase solvent. Theorganic solvent chosen should be capable of solubilizing sufficient drugand polymer to obtain a solution that can form microspheres when mixedwith a continuous phase, and subsequently one that can formmicroparticles upon removal of the solvent after dispersion of thediscontinuous phase in the continuous phase. For purposes ofsolubilizing sufficient quantities of both drug and polymer, acombination of solvents may be used. In particular embodiments, aquantity of a second solvent containing the drug is mixed with a solventcontaining the polymer. The second solvent is sufficiently miscible withthe first solvent, so that a clear, homogeneous solution is formed uponmixing the two discontinuous phase solvents. The second solvent may beimmiscible, partly miscible, or completely miscible with the continuousphase solvent.

The discontinuous phase solvent or solvent mixture may be immiscible, orpartly miscible with the continuous phase solvent. In particularembodiments employing a single discontinuous phase solvent, that solventmay be between 0.05% and 20% miscible with the continuous phase solvent.In another particular embodiment, the discontinuous phase solvent willbe between 1% and 10% miscible in the continuous phase solvent.

The discontinuous phase solvent is mixed with a continuous phase liquidcontaining appropriate emulsion stabilizers, as necessary to form anemulsion. The continuous phase liquid is typically not a solvent foreither the polymer or the encapsulated drug. Else continuous phase may,however, be a solvent for the discontinuous phase solvent or solvents.The volume or mass ratio of discontinuous phase solvent to continuousphase solvent during emulsification may be any ratio that allowsmicroparticles to be formed with the desired characteristics includingparticle size, and physical state of the drug encapsulated within themicroparticles, for example. In a particular embodiment, thediscontinuous phase to continuous phase volume ratios may range from0.5:1 to 30:1. In certain embodiments, the continuous phase may containfrom trace to saturating amounts of the discontinuous phase solvent or amixture of solvents designed to modulate the extraction of discontinuoussolvent or solvents from the oil phase of the emulsion. In a particularembodiment, the continuous phase liquid is water.

Emulsifiers may be added to the continuous phase liquid to stabilize theemulsion during formation and subsequent discontinuous phase solventremoval.

Examples of such emulsifiers include, phospholipids, such as lecithin,ionic and nonionic surfactants, poloxamers, or polymers such aspolyvinyl pyrrolidone and polyvinyl alcohol. In preferred embodiments,polyvinyl alcohol is used in concentrations ranging from 0.05 to 10%w/v. In a particularly embodiment, polyvinyl alcohol is used inconcentrations between 0.3 and 4% w/v in the aqueous phase.

In wet emulsification, particle size is controlled in part by the typeand amount of emulsifier contained in the aqueous phase, and also by themixing energy used to disperse the discontinuous phase into thecontinuous phase. Mixing may be effected by any of various meansincluding rapid stirring of the phases in a single vessel using amagnetic bar, impeller device, and rotor-statorhomogenizer, or probe orbath sonicators. In a particular embodiment, mixing is achieved bystirring with a magnetic bar.

Solvent removal from the discontinuous phase of the emulsion andconsequent hardening of microparticles may be achieved by various means.The emulsion may be held without extracting, at a predeterminedtemperature for a defined period of time prior to solvent extraction. Oralternatively, the organic solvent may be extracted immediately uponemulsification.

In certain embodiments of the present invention, solvent removal may becontrolled by evaporation. In other embodiments, the evaporation may beassisted by application of reduced pressure. In a certain embodiment,the solvent may be partly miscible with water, such that afteremulsification, rapid hardening of microparticles will result from theaddition of a sufficient further quantity of water to the emulsion tosolubilize all of the organic solvent contained in the emulsion. Therate at which the extraction medium is added to the emulsion, or therate at which the emulsion is added to the extraction medium may varydepending on the desired solvent extraction rate, which is in turndependent on the sensitivity of the drug/polymer system to changes insolution conditions. One of skill in the art is capable of determiningthese factors. In a further embodiment, discontinuous phase solventextraction may be achieved by modulating the temperature of the emulsionso that the solubility of the discontinuous phase in the continuousphase increases to sufficiently extract the discontinuous phase solvent.

EXAMPLES

It should be appreciated by those skilled in the art that the techniquesdisclosed in the examples which follow represent techniques discoveredby the inventors to function well in the practice of the invention, andthus can be considered to constitute the preferred modes for itspractice. However, those of skill in the art should appreciate, in lightof the present disclosure, that many changes can be made in the specificembodiments disclosed herein which will still obtain a like or similarresult without departing from the spirit and scope of the invention.

Example 1 Encapsulation of 2-methoxyestradiol intopoly-(lactide-co-glycolide) Microspheres

1.6 g of poly-(lactide-co-glycolide) (PLGA) with a 1:1 mole ratio oflactide to glycolide monomer and with an intrinsic viscosity of 0.15dl/g (PLGA 5050 2A, Medisorb, USA), and 800 mg 2-methoxyestradiol (2ME)were dissolved in 28 ml ethyl acetate by heating at 65° C. This oilphase was slowly poured into 80 ml of aqueous polyvinyl alcohol (av.Mol. Wt 100 kD, 1% w/v) in a 250 ml beaker containing a magnetic barstirring at 450 rpm. The mixture was thus emulsified for 5 min. beforethe emulsion was rapidly poured into 600 ml of 1% w/v aqueous polyvinylalcohol. The microspheres were allowed to harden for 3 hr. by magneticstirring at room temperature and ambient pressure. The hardenedparticles were collected and washed with water by centrifugation andthen lyophilized.

A sample of dry microspheres was dissolved in dimethyl sulfoxide (DMSO)and the 2ME present in the sample was quantified by HPLC analysisagainst standard concentrations of the drug. The core load was 28.0% 2MEby weight. Encapsulation efficiency was calculated as the percent ratioof measured core load to nominal core load, and was 85% for thispreparation.

18 mg 2ME microspheres, containing 5 mg 2ME, were suspended in a vehicleconsisting of 0.25 ml sodium carboxymethylcellulose, 2.5% by weight. Thesuspension was injected subcutaneously into Sprague Dawley rats. Threeanimals were sacrificed at specified time points, injection sites weredissected, and blood samples were withdrawn, and plasma was separatedand frozen at −80° C. Microsphere implants were isolated from theinjection sites and extracted with DMSO to quantity the unreleased drugby HPLC. The in vivo release was calculated by subtracting the amount ofdrug remaining in the implants from the total amount of 2ME injected.The in vivo release profile shows a burst release of approximately 35%in the first day, followed by linear release of 100% of the encapsulateddrug in 28 days.

The frozen plasma samples were thawed, extracted, derivatized, andplasma levels of 2ME were quantified by gas chromatography against known2ME standards. One day after injection, plasma 2ME levels were 6.5ng/nl, dropping to 5 ng/ml at day three. 2ME plasma concentrations thenincreased between day three and day seven, and were sustained at 8 ng/mlthrough day 14. Plasma concentrations of the drug then steadilydecreased between day 14 and day 28 to a final concentration of 2 ng/mlat day 28.

Example 2 Encapsulation of 2-hydroxyestradiol intopoly-(lactide-co-glycolide) Microspheres

A microsphere preparation was made by dissolving 1067 mg2-hydroxyestradiol (2HE) and 1600 mg PLGA (50:50 lactide:glycolide, Mw27 kD) in 28 ml ethyl acetate. The microspheres were prepared accordingto the details in Example 1. The core load was measured to be 38.3%, 96%encapsulation efficiency.

Microspheres equivalent to 5 mg 2HE were injected subcutaneously intorats. At predetermined intervals, animals were sacrificed and injectionsites were dissected to recover microspheres. At the same time, bloodsamples were taken from the animals, and the plasma was separated andfrozen at −80° C. The recovered microspheres were cleaned bycentrifugation and lyophilized. Carefully weighed samples were dissolvedin DMSO and the 2HE content of the recovered microspheres was quantifiedby HPLC analysis. Release of estradiol metabolites in vivo wascalculated indirectly by subtracting the amount of drug remaining inmicrospheres after in vivo incubation from the initial amount of drug inthe microspheres. 32% of the encapsulated drug was released within oneday of injection, with approximately 50% of the dose released afterthree days. The remaining drug was released steadily between day 3 andday 28 post-injection.

After the samples from each time point were collected, the frozen plasmasamples were thawed, extracted, derivatized, and plasma levels of 2HEand 2ME were quantified by gas chromatography against known standards.The plasma pharmacokinetic profile showed a blood level of approximately20 ng/ml 2HE, 24 hours post-injection, which decreased steadily to day 7post-injection. Blood levels of 2HE were detectable between trace levelsand 2 ng/ml between day 7 and day 28. A lower burst level of 2ME wasdetected (11.5 ng/ml) one day after injection, which trailed off throughday 7 to 2 ng/ml, which was sustained between day 7 and day 28.

Example 3 Encapsulation of 2HE in PLGA Microspheres by a SelectiveSolvent Extraction Method

Poly-D,L-(lactic-co-glycolic) acid in a 50:50 mole ratio (PLGA 50502.5M) with an average molecular weight of 27 kD was dissolved in ethylacetate to a concentration of 20% w/v. A second solution was made bydissolving 300 mg 2-hydroxyestradiol in 1.2 ml dimethyl sulfoxide()MSO). The two solutions were mixed by vortexing, resulting in asingle, clear solution. This organic solution was emulsified with anaqueous phase consisting of 17.5 ml water containing 700 mg polyvinylalcohol, 2.5 ml ethyl acetate, and 4 ml DMSO in a 50 ml beaker, bystirring with a 1 inch magnetic bar at 650 rpm for 5 min at 4° C. Theresulting emulsion was slowly poured into a 600 ml beaker containing 240ml water, 48 ml DMSO, and 6 ml ethyl acetate at 4° C. The particlesuspension was allowed to warm to room temperature under ambientconditions and ethyl acetate was allowed to extract/evaporate from theemulsion overnight. The hardened particles were filtered, washed withwater, and air dried prior to determining core load.

Dried microparticles from each preparation were solubilized in DMSO andquantified by HPLC against 2HE standards. The 2HE content was found tobe 13.1%. Encapsulation efficiency was 66%.

Microspheres equivalent to 5 mg 2HE were injected subcutaneously intorats. At predetermined intervals, animals were sacrificed and injectionsites were dissected to recover microspheres. At the same time, bloodsamples were taken from the animals, and the plasma was separated andfrozen at 80° C. The recovered microspheres were cleaned bycentrifugation and lyophilized. Carefully weighed samples were dissolvedin DMSO and the 2HE content of the recovered microspheres was quantifiedby HPLC analysis. Release of estradiol metabolites in vivo wascalculated indirectly by subtracting the amount of drug remaining inmicrospheres after in vivo incubation from the initial amount of drug inthe microspheres. In vivo release of 2HE was characterized by a burstrelease of 38% of the dose during the first day post-injection, followedby cumulative release of 55% at day 3 post-injection. 30% of the totaldose was released between day 3 and day 28, with a total of 88% of thedose released in 4 weeks.

After the samples from each time point were collected, the frozen plasmasamples were thawed, extracted, derivatized, and plasma levels of 2HEand 2ME were quantified by gas chromatography against known standards.The 38% burst release corresponded to a plasma level of 4 ng/ml 2HE,which decreased to trace levels between day 3 and day 28. Plasma levelsof 2ME peaked at 5.5 ng/ml one day post-injection, and decreased to 2ng/ml at day 3. This level was sustained between day 3 and day 28.

Example 4 Solid-In-Oil-In Water Encapsulation of 2ME

DMSO solutions of 2ME at a 25% w/y concentration were quench frozen inliquid nitrogen to prevent the crystallization of the drug. One mlaliquots of the frozen 2ME-DMSO solution were crushed in a cold mortarand pestle then mixed at 33,000 rpm with 10 ml of chilled 20% w/v PLGA50:50 (Oactide:glycolide, average Mw 53kD) solution in ethyl acetate,using a Fisher Powergen 125 rotor/stator homogenizer fitted with achilled 7 mm tip. Two preparations were made, the first was emulsifiedwith all components chilled to −20° C., and the second chilled to 4° C.Both temperatures maintained the 2ME-DMSO solution in the frozen state.The solid-in-oil emulsions were added to 30 ml distilled watercontaining 4% w/v polyvinyl alcohol and 3.5 ml ethyl acetate at 4° C.stirring at 600 rpm in a 100 ml beaker with a 1 inch stir bar. Theresulting solid-in-oil-in water emulsion was slowly poured into 250 mlice cold water in a 600 ml beaker containing 10 ml ethyl acetate, andstirred at 400 rpm with a 1.5 inch stir bar. The extraction beaker wasplaced into an ice bath during microsphere hardening. The beaker wasstirred overnight to extract the solvents from the microspheres, and thetemperature was allowed to increase to 22° C. slowly as the ice meltedand water warmed under ambient conditions.

The particles were collected by filtration, washed with water andlyophilized. Dried particles were dissolved in DMSO and the 2ME contentwas measured by HPLC. Encapsulation efficiency of the solid/oil/watertechnique at −20° C. and 4° C. is presented in Table I. TABLE I 2MEencapsulation efficiency using the solid-in-oil-in-water technique.Emulsification Nominal Measured Encapsulation Temperature, ° C. CoreLoad, % Core Load, % Efficiency, % −20 11.1 6.7 60 4 11.1 9.3 84

In vitro release of 2ME was measured by adding 12.5 mg microspheres to100 ml 50% aqueous ethyl alcohol. Aliquots were removed at predeterminedintervals and 2ME concentrations were measured by UV absorbancespectroscopy. The preparation that was emulsified at −20° C. released15% of the encapsulated drug in a linear fashion for 6 hours. Bycontrast the formulation that was emulsified at 4° C. released 7% of theencapsulated drug within 30 minutes, with the release rate steadilydecreasing between 30 minutes and 7 hours.

Example 5 Eutectic Mixture of 2ME and Menthol

For chronic administration of low dose drugs, a transdermal patch may bepreferable to repeated injections of PLGA microspheres. Two keyobstacles to transdermal administration are formulation of a stable,high concentration reservoir of drug, and some mechanism to enhance skinpermeability. Kaplun-Frischoff and Touitou (1997) J. Pharm. Sci.86:1394-1399 (Testosterone skin permeation enhancement by mentholthrough formation of eutectic with drug and interaction with skinlipids) found that testosterone formed a eutectic mixture with menthol,which is a known skin permeability enhancer. However, because 2ME has amuch lower solubility in alcohols than does testosterone, formation of aeutectic mixture of 2ME and menthol is not an obvious extension of thecurrent art.

187.6 mg of menthol was ground in a mortar and pestle with 121.3 mg 2MEfor five minutes, to form a homogeneous, waxy solid. 1.2 mg of this mitewas hermetically sealed in an aluminum pan and loaded into the samplecompartment of a TA Instruments Q10 differential scanning calorimeter at25° C. The sample was scanned from 25 to 160° C. at 10° C./min. Afirst-order endothermic peak was noted with an onset temperature of31.47° C. This peak does not correspond to either of the pure compounds(menthol 41.77° C., 2ME 187.36° C.), and is consistent with theformation of a separate, eutectic phase. Transdermal delivery of theestradiol metabolite is expected to be enhanced from the eutecticmixture with menthol, since the mixture will be liquid at bodytemperature.

Example 6 3-benzoyl-2-methoxyestradiol Microspheres Prepared by SprayDrying

536 mg 3-benzoyl-2-methoxyestradiol and 1250 mg PLGA (50:50lactide:glycolide, 27 kD Mw) were dissolved in 25 ml methylene chloride.The solution was pumped at 3 ml/min. through the inlet atomizer of aBuchi Mini Spray Dryer model B-191. Equipment settings were as follows:inlet temperature, 50° C.; outlet temperature, 43° C.; aspirator, 80%;atomizer air flow, 600 ml/min. Particles were collected and examined byscanning electron microscopy (06/18/02). Particle size ranged between0.2 and 3 um. No drug crystals were noted, indicating efficientencapsulation of the drug.

Example 7 2-methoxyestradiol Microspheres Containing an Antioxidant,Prepared by Extrusion Through a Packed-Bed Emulsifier

800 mg PLGA (50:50 lactide:glycolide, 13 kD average Mw), 400 mg2-methoxyestradiol, and 8.2 mg BHT were dissolved in 14 ml ethyl acetateat 65° C. This oil phase was emulsified with an aqueous phase consistingof 1% w/v polyvinyl alcohol at a ratio of 1 part oil to 1.5 parts water.The resulting emulsion was either pumped directly at 3 ml/min. into asolvent extraction medium consisting of 800 ml 0.5% w/v polyvinylalcohol, or held at 65° C. for approximately 10 min prior to adding theemulsion to the extraction medium. The microspheres were allowed toharden in the exaction medium by stirring at room temperature for 2hours. The hardened microspheres were collected by centrifugation,washed with distilled water, and lyophilized.

Dried microparticles from this preparation were solubilized in DMSO andquantified by HPLC against 2ME standards. The 2ME content was found tobe 33.2%. Encapsulation efficiency was 97%. BHT content of themicrospheres was quantified in the same assay against known BHTstandards. The BHT loading was 0.7% by weight, corresponding to 100%encapsulation efficiency for the preservative.

Example 8 2-methoxyestradiol Microspheres Prepared by TemperatureModulated Solvent Extraction

1600 mg PLGA (50:50 lactide:glycolide, 13 kD Mw) and 805 mg2-methoxyestradiol were dissolved in 28 ml ethyl acetate at 65° C. Thisoil phase was emulsified with an aqueous phase consisting of 1% w/vpolyvinyl alcohol at a ratio of 1 part oil to 20 parts water. Theresulting emulsion was pumped through a lag tube immersed in ice water.The solubility of ethyl acetate in water increases with decreasingtemperature, such that the cooled aqueous phase becomes a reservoir withincreasing capacity for the organic solvent. The cooled microspheresuspension was pumped into a beaker and the microspheres were hardenedby stirring at room temperature for 3 hours. The hardened microsphereswere collected by centrifugation, washed with distilled water, andlyophilized.

Dried microparticles from this preparation were solubilized in DMSO andquantified by HPLC against 2ME standards. The 2ME content was found tobe 27.3%. Encapsulation efficiency was 82%.

Example 9 Stabilization of 2-methoxyestradiol in Microspheres byAnnealing

Microspheres containing 2ME were prepared according to details inExample 6, above. The finished microsphere powder was mixed with a10-fold excess by weight of granular sucrose. The solid suspension wassealed in a polypropylene tube and immersed in a 65° C. water bath for 3hours to allow crystallization of the encapsulated 2-methoxyestradiol.After annealing, the tube was removed from the temperature bath andsufficient water was added to dissolve the sucrose. The microsphereswere washed three times with water by centrifugation, and lyophilized.Differential scanning calorimetry on a sample of the annealedmicrospheres confirmed that >99% of the drug had crystallized.

Samples of microspheres before and after annealing were loaded intoglass vials, sealed in ambient atmosphere, and placed into temperaturecontrolled incubators at 23° C. or 38° C. Vials were removed fromincubators at predetermined time intervals and the content and purity ofthe 2ME in the microspheres was analyzed. The formulation containingamorphous drug degraded by more than 13% when stored at roomtemperature, whereas the formulation containing crystalline drug did notdegrade during an incubation of 58 days at 38° C. TABLE II Storagestability of crystalline and amorphous 2ME in microspheres. 2MEcore load% crystalline 2ME, Lot # % w/w time zero Storage purity 050-049-SE 26.917 27 days, 23° C. 87.4% 041-158-B 30.4 99.7 58 days, 38° C. 99.3%

Example 10 2-methoxyestradiol Microspheres Prepared by Encapsulation ofMicronized Crystals

2-methoxyestradiol drug substance was ground in a mortar and pestleuntil the particles were less than 5 um diameter. Particle size wasmonitored by scanning electron microscopy. The micronized drug, 129 mg,was added to 1 ml of a 30% w/v solution of PLGA (50:50lactide:glycolide, 27 kD Mw) in ethyl acetate. The drug was suspended inthe polymer solution using a probe sonicator with the vessel placed onice. The suspension was added dropwise to 50 ml of aqueous 4% w/vpolyvinyl alcohol stirring at 800 rpm in a 150 ml beaker. Themicrosphere suspension was stirred overnight at room temperature. Thehardened microspheres were collected by centrifugation, washed withdistilled water, and lyophilized. Chromatographic analysis of a sampleof the final microsphere preparation dissolved in dimethyl sulfoxide,and measured against known 2ME standards, showed that the core load was28.8% 2-methoxyestradiol by weight, making the encapsulation efficiency96%.

Example 11 Preparation of 2-methoxyestradiol Transdermal PatchFormulations

2-methoxyestradiol drug substance was suspended in different transdermalgrade pressure sensitive adhesives (National Starch) and coated ontopolyethylene and aluminum vapor coated polyester backings (3M) using aGardco “Microm” film applicator. Coatings were dried overnight in a fumehood and drying completed in an 80° C. oven for 24 hours.

Example 12 Preparation of 2-methoxyestradiol-3,17-diacetate Microspheres

400 mg of poly-(lactide-co-glycolide) (PLGA) with a 1:1 mole ratio oflactide to glycolide monomer and with an intrinsic viscosity of 0.25dl/g, average Mw 27 kD (PLGA 5050 2.5M, Medisorb, USA), and 200 mg2-methoxyestradiol-3,17-diacetate were dissolved in 7 ml ethyl acetateby stirring at 23° C. This oil phase was slowly poured into 20 ml ofaqueous polyvinyl alcohol (av. Mol. Wt. 100 kD, 1% w/v) in a 50 mlbeaker containing a magnetic bar stirring at 450 rpm. The mixture wasthus emulsified for 5 min. before the emulsion was rapidly poured into150 ml of 1% w/v aqueous polyvinyl alcohol. The microspheres wereallowed to harden for 3 hr. by magnetic stirring at room temperature andambient pressure. The hardened particles were collected and washed withwater by centrifugation and then lyophilized.

A second preparation was made by dissolving 400 mg each of the PLGA and2-methoxyestradiol-3,17-diacetate in 7 ml ethyl acetate. Emulsificationwas performed as described above.

A sample of dry microspheres from each preparation was dissolved indimethyl sulfoxide (DMSO) and the 2ME diacetate present in the sampleswas quantified by HPLC analysis against standard concentrations of thedrug. Drug loading and encapsulation efficiency are detailed in TableIII. TABLE III Drug loading for 2-methoxyestradiol-3,17-diacetatemicrospheres. Nominal Measured Encapsulation Lot # Loading LoadingEfficiency 041-034-A 33.3% 29.8% 89% 041-034-B 50.0% 44.1% 88%

Microspheres from each preparation equivalent to 5 mg 2ME diacetate wereadded to 100 ml of 50% aqueous alcohol. The vessels were stirred at 100rpm at 23° C. for 7 hours. Samples of the release medium were withdrawnat intervals, and the concentration of the drug was measured by UVabsorbance at 287 nm. Lot 041-034-A released 5% of the encapsulated drugin 1 hr, with 8% total release in 7 hours in the in vitro release assay.In contrast, 8% of the encapsulated drug was released within 30 min.from lot 041-034-B, with a subsequent total of 24% released in 7 hours.

Microspheres equivalent to 5 mg 2ME diacetate from lot # 041-034-Bdescribed above, were injected subcutaneously into Sprague Dawley rats.Blood was collected periodically over 4 weeks. Immediately aftercollection, plasma was separated and stored frozen at −80° C. After thesamples from each time point were collected, the frozen plasma sampleswere thawed, extracted, derivatized, and plasma levels of 2ME werequantified by gas chromatography against known 2ME standards. Thepharmacokinetic profile was characterized by a steady increase to 2ng/ml between day 0 and day3. Plasma levels were sustained between 1 and3 ng/ml between day 3 and day 28. Interestingly, there was no largeburst release of 2-methoxyestradiol observed during the first three daysafter injection from microsphere formulations containing the diacetateprodrug.

All of the COMPOSITIONS, METHODS and APPARATUS disclosed and claimedherein can be made and executed without undue experimentation in lightof the present disclosure. While the compositions and methods of thisinvention have been described in terms of preferred embodiments, it willbe apparent to those of skill in the art that variations may be appliedto the COMPOSITIONS, METHODS and APPARATUS and in the steps or in thesequence of steps of the methods described herein without departing fromthe concept, spirit and scope of the invention. More specifically, itwill be apparent that certain agents that are both chemically andphysiologically related may be substituted for the agents describedherein while the same or similar results would be achieved. All suchsimilar substitutes and modifications apparent to those skilled in theart are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

1. A composition of matter comprising: (a) an estradiol metabolite; and(b) a material providing for sustained release.
 2. The composition ofclaim 1, wherein said material providing for sustained release isselected from the group consisting of microparticles, nanoparticles,patches, crystals, gels, rods, stints, pellets, discs, lozenges, wafers,capsules, films, microcapsules, nanocapsules, hydrogels, liposomes,implants and vaginal rings.
 3. The composition of claim 2, wherein saidmicroparticles or nanoparticles are comprised of a biodegradable polymerselected from the group consisting of poly(lactide)s, poly(glycolide)s,poly(lactide-co-glycolide)s, poly(lactic acid)s, poly(glycolic acid)s,poly(lactic acid-co-glycolic acid)s, polycaprolactone, polycarbonates,polyesteramides, polyanhydrides, poly(amino acids), polyorthoesters,polyacetyls, polycyanoacrylates, polyetheresters, poly(dioxanone)s,poly(alkylene alkylate)s, copolymers of polyethylene glycol andpolyorthoester, polyurethanes, blends and copolymers thereof.
 4. Thecomposition of claim 3, wherein said microparticles or nanoparticles arecomprised of poly(d,l-lactide-co-glycolide).
 5. The composition of claim2, wherein said microparticles have a diameter between 1 and 200micrometers.
 6. The composition of claim 2, wherein said nanoparticleshave a diameter between 20 and 2000 nanometers.
 7. The composition ofclaim 2, wherein said microparticles or nanoparticles further comprisean additive.
 8. The composition of claim 7, wherein said additive isselected from the group consisting of butylated hydroxytoluene, propylgallate, a α-tocopherol, ascorbyl palmitate, an antioxidant, a releasemodifier and a buffer.
 9. The composition of claim 1, wherein saidestradiol metabolite is selected from the group consisting of 2-methoxyestradiol, 2-hydroxy estradiol, 4-methoxy estradiol and 4-hydroxyestradiol.
 10. The composition of claim 1, wherein said estradiolmetabolite is delivered transmucosally.
 11. The composition of claim 10,wherein said transdermal transmucosal delivery is selected from thegroup consisting of buccal, oral, ocular, nasal, rectal and vaginal. 12.The composition of claim 1, wherein said estradiol metabolite is aprodrug.
 13. The composition of claim 12, wherein said prodrug is anester.
 14. The composition of claim 13, wherein said ester is selectedfrom the group consisting of 3-benzoyl-2-methoxy estradiol;17-benzoyl-2-methoxy estradiol; 17-acetyl-2-methoxy estradiol;3-acetyl-2-methoxy estradiol; 3,17-dibenzoyl-2-methoxy estradiol;3,17-diacetyl-2-methoxy estradiol; 3-benzoyl-4-methoxy estradiol;17-benzoyl-4-methoxy estradiol; 17-acetyl-4-methoxy estradiol;3-acetyl-4-methoxy estradiol; 3,17-dibenzoyl-4-methoxy estradiol;3,17-diacetyl-4-methoxy estradiol; 3-benzoyl -2-hydroxy estradiol;17-benzoyl-2-hydroxy estradiol; 17-acetyl-2-hydroxy estradiol;3-acetyl-2-hydroxy estradiol; 3,17-dibenzoyl-2-hydroxy estradiol;3,17-diacetyl-2-hydroxy estradiol; 2,3-dibenzoyl-2-hydroxy estradiol;2,17-dibenzoyl-2-hydroxy estradiol; 2,17-diacetyl-2-hydroxy estradiol;2,3-diacetyl-2-hydroxy estradiol; 2,3,17-tribenzoyl-2-hydroxy estradiol;2,3,17-triacetyl-2-hydroxy estradiol; 3-benzoyl-4-hydroxy estradiol;17-benzoyl-4-hydroxy estradiol; 17-acetyl-4-hydroxy estradiol;3-acetyl-4-hydroxy estradiol; 3,17-dibenzoyl-4-hydroxy estradiol;3,17-diacetyl-4-hydroxy estradiol; 3,4-dibenzoyl-4-hydroxy estradiol;4,17-dibenzoyl-4-hydroxy estradiol; 4,17-diacetyl-4-hydroxy estradiol;3,4-diacetyl-4-hydroxy estradiol; 3,4,17-tribenzoyl-4-hydroxy estradiol;3,4,17-triacetyl-4-hydroxy estradiol.
 15. The composition of claim 1,wherein said estradiol metabolite is in a eutectic mixture.
 16. Thecomposition of claim 1, further comprising a hydrophilic polymer. 17.The composition of claim 16, wherein said hydrophilic polymer isselected from the group consisting of poly(ethylene glycol),poly(propylene glycol) and copolymers of poly(ethylene glycol) andpoly(propylene glycol).
 18. The composition of claim 1, wherein saidestradiol metabolite is derivatized.
 19. The composition of claim 18,wherein said derivative is selected from the group consisting ofdicarboxylic acid compounds, diacids, polar compounds and ioniccompounds.
 20. The composition of claim 19, wherein such dicarboxylicacid compound is selected from the group consisting of oxalic, malonic,maleic, succinic, glutaric, adipic, pimelic and pamoic acid.
 21. Thecomposition of claim 19, wherein such diacids are selected from thegroup consisting of succinic, glutaric, maleic, malonic and oxalic acid.22. (canceled)
 23. (canceled)
 24. (canceled)
 25. (canceled) 26.(canceled)
 27. (canceled)
 28. (canceled)
 29. (canceled)
 30. (canceled)31. (canceled)
 32. (canceled)
 33. (canceled)
 34. The composition ofclaim 1, wherein the composition is useful to treat an individual.
 35. Atreatment method comprising: (a) administering to an individual asustained release formulation containing an estradiol metabolite. 36.The method of claim 35, wherein said sustained release formulationcontaining an estradiol metabolite is selected from the group consistingof microparticles, nanoparticles, patches, crystals, gels, rods, stints,pellets, discs, lozenges, wafers, capsules, films, microcapsules,nanocapsules, hydrogels, liposomes, implants and vaginal rings.
 37. Themethod of claim 36, wherein said microparticles or nanoparticles arecomprised of a biodegradable polymer selected from the group consistingof poly(lactide)s, poly(glycolide)s, poly(lactide-co-glycolide)s,poly(lactic acid)s, poly(glycolic acid)s, poly(lactic acid-co-glycolicacid)s, polycaprolactone, polycarbonates, polyesteramides,polyanhydrides, poly(amino acids), polyorthoesters, polyacetyls,polycyanoacrylates, polyetheresters, poly(dioxanone)s, poly(alkylenealkylate)s, copolymers of polyethylene glycol and polyorthoester,biodegradable polyurethanes, blends and copolymers thereof.
 38. Themethod of claim 37, wherein said microparticles or nanoparticles arecomprised of poly(d,l-lactide-co-glycolide).
 39. The method of claim 37,wherein said microparticles have a diameter between 1 and 200micrometers.
 40. The method of claim 37, wherein said nanoparticles havea diameter between 20 and 2000 nanometers.
 41. The method of claim 37,wherein said microparticles or nanoparticles further comprise anadditive.
 42. The method of claim 41, wherein said additive is selectedfrom the group consisting of butylated hydroxytoluene, propyl gallate,α-tocopherol, ascorbyl palmitate, an antioxidant, a release modifier anda buffer.
 43. The method of claim 35, wherein said estradiol metaboliteis selected from the group consisting of 2-methoxy estradiol, 2-hydroxyestradiol, 4-methoxy estradiol and 4-hydroxy estradiol.
 44. The methodof claim 35, wherein said estradiol metabolite is deliveredtransmucosally.
 45. The method of claim 44, wherein said transmucosaldelivery is selected from the group consisting of buccal, oral, ocular,nasal, rectal and vaginal.
 46. The method of claim 35, wherein saidestradiol metabolite is a prodrug.
 47. The method of claim 46, whereinsaid prodrug is an ester.
 48. The method of claim 47, wherein said esteris selected from the group consisting of 3-benzoyl-2-methoxy estradiol;17-benzoyl-2-methoxy estradiol; 17-acetyl-2-methoxy estradiol;3-acetyl-2-methoxy estradiol; 3,17-dibenzoyl-2-methoxy estradiol;3,17-diacetyl-2-methoxy estradiol; 3-benzoyl-4-methoxy estradiol;17-benzoyl-4-methoxy estradiol; 17-acetyl-4-methoxy estradiol;3-acetyl-4-methoxy estradiol; 3,17-dibenzoyl-4-methoxy estradiol;3,17-diacetyl-4-methoxy estradiol; 3-benzoyl -2-hydroxy estradiol;17-benzoyl-2-hydroxy estradiol; 17-acetyl-2-hydroxy estradiol;3-acetyl-2-hydroxy estradiol; 3,17-dibenzoyl-2-hydroxy estradiol;3,17-diacetyl-2-hydroxy estradiol; 2,3-dibenzoyl-2-hydroxy estradiol;2,17-dibenzoyl-2-hydroxy estradiol; 2,17-diacetyl-2-hydroxy estradiol;2,3-diacetyl-2-hydroxy estradiol; 2,3,17-tribenzoyl-2-hydroxy estradiol;2,3,17-triacetyl-2-hydroxy estradiol; 3-benzoyl-4-hydroxy estradiol;17-benzoyl-4-hydroxy estradiol; 17-acetyl-4-hydroxy estradiol;3-acetyl-4-hydroxy estradiol; 3,17-dibenzoyl-4-hydroxy estradiol;3,17-diacetyl-4-hydroxy estradiol; 3,4-dibenzoyl-4-hydroxy estradiol;4,17-dibenzoyl-4-hydroxy estradiol; 4,17-diacetyl-4-hydroxy estradiol;3,4-diacetyl-4-hydroxy estradiol; 3,4,17-tribenzoyl-4-hydroxy estradiol;3,4,17-triacetyl-4-hydroxy estradiol.
 49. The method of claim 35,wherein said sustained release formulation containing an estradiolmetabolite is in a eutectic mixture.
 50. The method of claim 35, furthercomprising a hydrophilic polymer.
 51. The method of claim 50, whereinsaid hydrophilic polymer is selected from the group consisting ofpoly(ethylene glycol), poly(propylene glycol) and copolymers ofpoly(ethylene glycol) and poly(propylene glycol).
 52. The method ofclaim 35, wherein said estradiol metabolite is derivatized.
 53. Themethod of claim 52, wherein said derivative is selected from the groupconsisting of dicarboxylic acid compounds, diacids, polar compounds andionic compounds.
 54. The method of claim 53, wherein such dicarboxylicacid compound is selected from the group consisting of oxalic, malonic,maleic, succinic, glutaric, adipic, pimelic and pamoic acid.
 55. Themethod of claim 53, wherein such diacids is selected from the groupconsisting of succinic, glutaric, maleic, malonic and oxalic acid. 56.The method of claim 35, wherein said sustained release formulationcontaining an estradiol metabolite is produced by spray drying asolution of polymer and estradiol metabolite dissolved in an organicsolvent.
 57. The method of claim 35, wherein said sustained releaseformulation containing an estradiol metabolite is produced by wetemulsification including a continuous and discontinuous phase followedby solvent removal.
 58. The method of claim 57, wherein saiddiscontinuous phase contains estradiol metabolites and polymer.
 59. Themethod of claim 58, further comprising an additive.
 60. The method ofclaim 59, wherein said additive is selected from the group consisting ofan antioxidant, a buffer, and a release modifier.
 61. The method ofclaim 57, wherein said discontinuous phase contains an organic solvent.62. The method of claim 61, wherein said organic solvent is selectedfrom the group consisting of one solvent, two solvents and a mixture ofsolvents.
 63. The method of claim 57, wherein said continuous phasefurther comprises an emulsifier.
 64. The method of claim 63, whereinsaid emulsifier is selected from the group consisting of phospholipids,lecithin, ionic surfactants, nonionic surfactants, poloxamers, polymers,polyvinyl pyrrolidone and polyvinyl alcohol.
 65. The method of claim 64,wherein said emulsifier is polyvinyl alcohol.
 66. The method of claim35, wherein said sustained release formulation containing an estradiolmetabolite was produced by the selective extraction of an oil phasesolvent.
 67. (canceled)
 68. A composition of matter comprising; (a) anestradiol metabolite selected from the group consisting ofcatecholestrogens and methoxyestradiols; and (b) a material providingfor sustained release.
 69. The composition of claim 68, wherein saidmaterial providing for sustained release is a microparticle ornanoparticle comprised of a biodegradable polymer selected from thegroup consisting of poly(lactide)s, poly(glycolide)s,poly(lactide-co-glycolide)s, poly(lactic acid)s, poly(glycolic acid)s,poly(lactic acid-co-glycolic acid)s, polycaprolactone, polycarbonates,polyesteramides, polyanhydrides, poly(amino acids), polyorthoesters,polyacetyls, polycyanoacrylates, polyetheresters, poly(dioxanone)s,poly(alkylene alkylate)s, copolymers of polyethylene glycol andpolyorthoester, biodegradable polyurethanes, blends and copolymersthereof.
 70. The composition of claim 68, wherein said estradiolmetabolite is selected from the group consisting of 2-methoxy estradiol,2-hydroxy estradiol, 4-methoxy estradiol and 4-hydroxy estradiol.